Propiconazole resistant mutants of Chlorella species

ABSTRACT

The present disclosure relates to propiconazole resistant mutants of Chlorella sp. having Accession No. CCAP 211/133. The propiconazole resistant mutants of Chlorella species have increased tolerance to propiconazole. The present disclosure further provides a method for preparing the propiconazole resistant mutants of Chlorella species. The propiconazole resistant mutants of Chlorella species can selectively grow in the presence of propiconazole, and hence can be used for large scale production of algal biomass.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application which claims priorityto Indian Patent Application No. 201621019072, filed on 2 Jun. 2016, thecontents of which are hereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to propiconazole resistant mutants ofChlorella species.

DEFINITIONS

As used in the present disclosure, the following terms are generallyintended to have the meaning as set forth below, except to the extentthat the context in which they are used indicate otherwise.

Grazers: The term “grazers” refer to aquatic pests such as, rotifers,dinoflagellates, ciliates, protozoans and fungi, which decrease theyield of algal biomass/production.

Contaminating organisms: The term “contaminating organisms” refers tofilamentous and unicellular cyanobacteria, flagellates, diatoms, etc.which compete with algae in utilization of resources and subsequentlyresult in decrease of algal biomass.

Algae: The term “algae” refers to an informal term for a large, diversegroup of eukaryotes that are not necessarily closely related and arethus polyphyletic. Included organisms range from unicellular genera,such as Chlorella to multicellular forms, such as the giant kelp, alarge brown alga that may grow up to 50 meters in length.

Propiconazole: Propiconazole is a triazole fungicide, also known as aDMI, or demethylation inhibiting fungicide due to its binding with andinhibiting the 14-alpha demethylase enzyme from demethylating aprecursor to ergosterol. Without this demethylation step, theergosterols are not incorporated into the growing fungal cell membranes,and cellular growth is stopped.

BACKGROUND

A major challenge in obtaining large quantities of algal biomass at avery low cost is to attain a continuous and stable production of algae.To achieve this, the algae have to be stably cultivated round the yearin open ponds at a sufficiently high density. However, algae whencultivated in open ponds are prone to numerous abiotic and bioticstresses, which lead to frequent culture crashes and productivitylosses. Abiotic stress can be taken care to a large extent through bioprospecting, screening and selection of the right kind of algal strainsfor a particular geographic location and season. Managing contaminantsin open ponds along with maintaining high productivity of algae forsuccessful biofuel production is very difficult.

Grazers and contaminating organisms are a major concern in thecultivation of algae. Ciliates, dinoflagellates, rotifers, diatoms,filamentous and unicellular cyanobacteria, flagellates, etc, either feedor compete with the algae for nutrients, resulting in reduced productionof the algal biomass. Various chemicals are known, which can be used tokill the grazers and the contaminating organisms, however, thesechemicals also have a deleterious effect on the algal growth.

Therefore, there is felt a need for large scale production of algae thatmitigates the drawbacks mentioned hereinabove.

OBJECTS

Some of the objects of the present disclosure, which at least oneembodiment herein satisfies, are as follows:

It is an object of the present disclosure to ameliorate one or moreproblems of the prior art or to at least provide a useful alternative.

An object of the present disclosure is to provide propiconazoleresistant mutants of Chlorella sp.

Another object of the present disclosure is to provide a method forpreparing propiconazole resistant mutants of Chlorella sp.

Still another object of the present disclosure is to providepropiconazole resistant mutants of Chlorella sp. exhibiting enhancedcrop protection, increased biomass, using propiconazole resistanceselection.

Still another object of the present disclosure is to provide a methodfor selectively improving the yield of Chlorella sp. by controllinggrazers and contaminating organisms in an aquatic environment, usingpropiconazole resistant mutants of Chlorella sp.

Other objects and advantages of the present disclosure will be moreapparent from the following description, which is not intended to limitthe scope of the present disclosure.

SUMMARY

The present disclosure relates to propiconazole resistant mutants ofChlorella sp. In one aspect, the propiconazole resistant mutants ofChlorella sp. have the characteristics of The Culture Collection ofAlgae and Protozoa (CCAP) Accession No. CCAP 211/133, having anincreased tolerance to propiconazole. The increase in tolerance isdefined relative to increase in biomass productivity using thepropiconazole resistant mutants of Chlorella sp. wherein the increase inbiomass productivity is in the range of 10% to 30%.

The propiconazole resistant mutants of Chlorella sp. having AccessionNo. CCAP 211/133 is prepared by subjecting the Chlorella sp. cells to UVradiation, reviving the UV treated Chlorella culture and treating therevived Chlorella culture with a pre-determined concentration ofpropiconazole, to obtain strains of propiconazole resistant mutants ofChlorella sp. resistant to propiconazole. The strains are then separatedand each of the strains is separately cultured in a culture media toidentify and obtain propiconazole resistant mutants of Chlorella sp.strains of exhibiting increase in biomass productivity in the range of10% to 30%, having Accession No. CCAP 211/133.

In a further aspect of the present disclosure, there is provided amethod for selectively controlling the growth of grazers in an aquaticenvironment. The method includes inoculating the aquatic environmentwith propiconazole resistant mutants of Chlorella sp. having AccessionNo. CCAP 211/133. The aquatic environment is treated with an effectiveconcentration of propiconazole to selectively control the growth ofgrazers. Propiconazole can be applied either before inoculating theaquatic environment with the propiconazole resistant mutants ofChlorella sp., or after inoculating the aquatic environment with thepropiconazole resistant mutants of Chlorella sp., or both before andafter inoculating the aquatic environment with the propiconazoleresistant mutants of Chlorella sp.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWING

The disclosure will now be described with reference to the accompanyingnon-limiting drawing, wherein:

FIG. 1 represents a graph illustrating the growth of wild type Chlorellasorokiniana and propiconazole resistant mutants of Chlorella sp. havingAccession No. CCAP 211/133, in the presence of propiconazole.

DETAILED DESCRIPTION

Algae are a source of various chemicals/compounds having usefulcommercial applications, such as, generation of renewable energy likebio-fuel which is an alternative to liquid fossil fuels. The use ofalgae as an alternative for production of bio-oils requires large scaleproduction of the algae. However, controlling the contaminants in openponds along with maintaining high productivity of algae for successfulbiofuel production is very difficult. The technologies used to managesuch competition or predation in open ponds have proved to beunsuccessful as these methods limit the desired productivity or areincompatible with the algal strain of interest or are unable to actagainst a wide variety of grazers and contaminating organisms or areeconomically unviable.

Various chemicals are known, which can be used to kill the grazers andthe contaminating organisms, however, these chemicals also have adeleterious effect on the algal growth. Propiconazole is a chemicalhaving a very potent activity against grazers and contaminatingorganisms. However, propiconazole is algicidal and hence harms the algaeas well. The use of chemicals, such as, propiconazole as a cropprotection agent in algal cultivation would therefore require algalstrains whose growth is not impacted by the presence of propiconazole.

Individual mutants are then isolated and screened to obtain thepropiconazole resistant algal mutants of the present disclosure.

Propiconazole is used for screening and selecting algal mutants as it isan effective grazicide at a very low dose. Propiconazole is a potentgrazicide at 0.5 ppm and above. It also acts as an endogenous inhibitorof Brassinosteroid (BR) synthesis in plants. BR inhibitors likepropiconazole target the inhibition of key enzymes like Cytochrome P450monooxygenase of BR biosynthetic pathway. It is suggested that thetriazole inhibitors like propiconazole inhibit the hydroxylation of theC-22 position of the side chain in BRs by directly binding to DWF4.DWF4, is a cytochrome P450 isolate of putative steroid 22-hydroxylase.The production of Brassinosteroids (BRs) is known and quantified inaxenic microalgae strains belonging to Chlorophyceae, Trebouxiophyceae,Ulvophyceae, and Charophyceae family. Two brassinosteroids, viz,brassinolide and castasterone are found in all the algal strains. BRproduction in Chlorella sp. is suggested to enhance cell proliferation,protein and metabolite content.

Accordingly, the present disclosure envisages propiconazole resistantmutants of Chlorella sp. that are able to grow in the presence ofpropiconazole at concentrations that are otherwise toxic to the wildtype algae. The ability of the propiconazole resistant mutants ofChlorella sp. to grow on propiconazole concentrations that are lethal tothe various grazers and contaminating organisms further provide a cropprotection benefit for the mutants. These propiconazole resistantmutants of Chlorella sp. can yield higher biomass.

The present disclosure also provides a method for preparingpropiconazole resistant mutants of Chlorella sp. The method includesgenerating, selecting, isolating and characterizing propiconazoleresistant mutants of Chlorella sp. Typically, the mutation of the wildtype algae can be carried out by UV mutation, gamma mutation, andchemical mutation. In an embodiment of the present disclosure, themutation of the wild type algae is carried out by UV mutation. Aftermutation, the algal mutants are subsequently selected and enriched bygrowing the algal mutants for many generations in the presence ofpropiconazole.

In accordance with one aspect of the present disclosure, there isprovided propiconazole resistant mutants of Chlorella sp. having thecharacteristics of The Culture Collection of Algae and Protozoa (CCAP)Accession No. CCAP 211/133. The propiconazole resistant mutants ofChlorella sp. have an increased tolerance to propiconazole.

The increase in tolerance is defined relative to increase in biomassproductivity using the propiconazole resistant mutants of Chlorella sp.Typically, the increase in biomass productivity is in the range of 10%to 30%.

The present disclosure provides a method for preparing the propiconazoleresistant mutants of Chlorella sp. having Accession No. CCAP 211/133.

The method comprises subjecting wild type Chlorella sp. cells to UVradiation to obtain a UV treated Chlorella culture. In an embodiment ofthe present disclosure, the wild type Chlorella sp. is Chlorellasorokiniana, and can be sourced from Gagva, Jamnagar, Gujarat, India.Typically, the UV dose can be in the range of 200 mJ/m² to 700 mJ/m².Typically, the wild type Chlorella sp. is subjected to the aforestatedUV dose for a time period in the range of 15 seconds to 120 seconds. Thecells are distributed as a monolayer to ensure uniform and efficientpenetration of UV. In an embodiment, a UV crosslinker is used for the UVradiation.

The UV treated Chlorella culture is then revived to obtain a revivedChlorella culture. The cells are typically revived in 12 h light/12 hourdark cycles of white light on culture racks, over a time period in therange of 12 days to 14 days.

The revived Chlorella culture is treated with a pre-determinedconcentration of propiconazole, to obtain strains of propiconazoleresistant mutants of Chlorella sp., which are resistant topropiconazole. The pre-determined concentration of propiconazole can bein the range of 5 μM to 15 μM. In one embodiment, the concentration ofpropiconazole is 10 μM. In another embodiment, the revived UV treatedpropiconazole resistant mutants of Chlorella sp. is passed through 30generations in the presence of 10 μM propiconazole, to enrich the mutantpool.

The strains of propiconazole resistant mutants of Chlorella sp. areseparated and each strain is separately cultured in a culture media toidentify and obtain strains of propiconazole resistant mutants ofChlorella sp. that exhibit increase in biomass productivity in the rangeof 10% to 30%, having Accession No. 211/133.

In one embodiment, the media is urea-phosphoric acid medium.

The present disclosure further provides a method for selectivelycontrolling the growth of grazers in an aquatic environment. The aquaticenvironment can be fresh water or sea water, and can be selected fromthe group consisting of outdoor pond, tank for commercial aquaculture,pond for commercial aquaculture, aquarium, and photo-bioreactor.Typically, the pH of the aquatic environment can be in the range of 6.0to 10.0.

The aquatic environment is inoculated with propiconazole resistantmutants of Chlorella species having Accession No. CCAP 211/133. Theaquatic environment is treated with an effective concentration ofpropiconazole, to selectively control the growth of grazers.

In an embodiment, the treatment with is carried out before inoculatingthe aquatic environment with the propiconazole resistant mutants ofChlorella species.

In another embodiment, the treatment with propiconazole is carried outafter inoculating the aquatic environment with the propiconazoleresistant mutants of Chlorella species.

In still another embodiment, the treatment with propiconazole is carriedout both before, and after inoculating the aquatic environment with thepropiconazole resistant mutants of Chlorella species.

Typically, the concentration of propiconazole is in the range of 5 μM to15 μM.

Adding propiconazole to aquatic environment will kill the grazers,thereby increasing the yield of algae, i.e., propiconazole resistantmutants of Chlorella species, and hence selectively improves the yieldof the propiconazole resistant mutants of Chlorella species in anenvironment. Propiconazole exhibits very potent activity against grazersand contaminating organisms, and at the same time is a potent algicide.However, the propiconazole resistant mutants of Chlorella species of thepresent disclosure are capable of growing in the presence ofpropiconazole and their growth is not impacted by the presence ofpropiconazole.

The propiconazole resistant mutants of Chlorella species of the presentdisclosure can grow in the presence of propiconazole at concentrationsthat kills the wild type algae. The ability of the propiconazoleresistant mutants of Chlorella species to grow in the presence ofpropiconazole provides protection from grazers and contaminatingorganisms. Further, some of the propiconazole resistant mutants ofChlorella species are surprisingly found to have higher biomass.

The present disclosure is further described in light of the followinglaboratory scale experiments which are set forth for illustrationpurpose only and not to be construed for limiting the scope of thedisclosure. These laboratory scale experiments can be scaled up toindustrial/commercial scale and the results obtained can be extrapolatedto industrial/commercial scale.

EXPERIMENTAL DETAILS Experiment-1: Preparation of PropiconazoleResistant Mutants of Chlorella Species in Accordance with the PresentDisclosure

Urea-Phosphoric acid medium having the composition as summarized inTable-1 was used for all the studies.

TABLE 1 Composition if Urea - Phosphoric acid medium Ingredient AmountDistilled water 900 ml Instant ocean salts* 42 gm/L (gives salinity of4%) Urea 3.3 mM Phosphoric acid (H₃PO₄) 0.21 mM Trace elements finalconcentration below FeCl₃ 23.4 μM EDTA 23.4 μM CuSO₄ 5H₂O 78.6 nMNa₂MoO₄ 2H₂O 52 nM ZnSO₄ 7H₂O 0.153 μM CoCl₂ 6H₂O 84 nM MnCl₂ 4H₂O 1.82μM Adjust pH to 7.5 and make volume to 1 L using distilled water*Instant Ocean was procured from Aquarium systems Inc. USA.

Step-I: UV Mutation Procedure

1×10⁷ Cells/mL wild type Chlorella sorokiniana cells (Gagva, Jamnagar,Gujarat, India) at log phase of growth (O.D₇₅₀ 0.5-0.7) were taken andcentrifuged at 5000 rpm for 10 minutes at 30° C. to obtain a pellet. Thepellet was re-suspended in 10 ml of Urea-Phosphoric acid medium andplaced on a glass petri plate (autoclaved) without lid. Care was takento ensure that the cells formed a monolayer on the glass plate, so as toaid in efficient and uniform UV penetration. The cells were exposed toUV radiation in UV crosslinker for different times (30 seconds to 90seconds) depending on the UV dose (300-500 mJ/m²). After completion ofthe UV exposure, the cells were collected into a 50 ml tube and kept indark for 24 hours. After 24 hours, the cells were centrifuged at 5000rpm for 10 minutes at room temperature and re-suspended in 5 ml ofUrea-Phosphoric acid medium in culture tube.

Cell viability of Untreated (control) and UV exposed cells wascalculated using nucleic acid dye (Sytox green). 0.5 μM of the dye wasused for staining and incubated for 5 minutes in the dark. The cellswere analyzed on flow cytometer. Killing was about 40-60% using UVradiation. Cells were revived in 12 h light/12 hour dark cycles of whitelight on culture racks. The revival of the cells took 12-14 days.

Step-II: Enrichment of Propiconazole Resistant Mutants of ChlorellaSpecies

UV mutagenized and revived cells were exposed to sub lethalconcentration of propiconazole (10 μM). To enrich the mutant pool, theUV mutagenized culture was passed through 30 generations in the presenceof 10 μM propiconazole.

Step-III: Separation, Isolation and Identification of IndividualPropiconazole Resistant Mutants of Chlorella Strains

The enriched mutant pool was plated on 0.8% agar plates containing 10 μMpropiconazole. About 350 isolated colonies were obtained. Theseindividual propiconazole resistant mutants of Chlorella speciesresistant to propiconazole were patched on 0.8% agar plates containing10 μM propiconazole. These mutants were then inoculated in 2 mL ofsterile liquid Urea-Phosphoric acid medium in culture tubes. This wasscaled up to 10 mL of sterile liquid Urea-Phosphoric acid medium in 50mL flasks. The growth kinetics of individual mutants was carried outwith and without 10 μM propiconazole in 250 mL flask using 70 mL medium.

Strains of propiconazole resistant mutants of Chlorella speciesexhibiting increase in biomass productivity in the range of 10% to 30%were identified and deposited at the Culture Collection of Algae andProtozoa (CCAP), SAMS Limited, Scottish Marine Institute, OBAN, ArgyllPA37 1QA, Scotland, United Kingdom, having the Accession No. CCAP211/133 (Chlorella sorokiniana PcZ-1).

Experiment-2: Study of Wild Type Chlorella sorokiniana and PropiconazoleResistant Mutants of Chlorella Species in the Presence of Propiconazole

Chlorella sorokiniana cells (Wild type) and propiconazole resistantmutants of Chlorella species (PcZ-1) were inoculated at 0.1-0.15 OD@750nm (each set in triplicates). To verify the effect of methanol(propiconazole was dissolved in methanol), one set was treated with 0.1%methanol. One set of flasks in triplicate of Wild type and PcZ-1 mutantswere inoculated with and without 10 μM propiconazole. After inoculationflasks were kept in 12 h light/12 hour dark cycles of white light in anincubator shaker at 100 rpm. The temperature was 28° C., 70% humidityand 2% ambient CO₂. 3 mL sample was removed each day, to determine theOptical density (OD) of the culture at 750 nm. The results obtained areas illustrated in FIG. 1.

FIG. 1 illustrates a graph of the growth comparison between wild typeChlorella sorokiniana and propiconazole resistant mutants of Chlorellaspecies Chlorella sorokiniana mutant PcZ-1, in the presence ofpropiconazole, wherein A represents wild type Chlorella sorokinianagrown in the absence of propiconazole, B represents wild type Chlorellasorokiniana grown in the presence of 0.1% methanol, C represents wildtype Chlorella sorokiniana grown in the presence of 10 μM propiconazole,D represents PcZ-1 grown in the absence of propiconazole, E representsPcZ-1 grown in the presence of 0.1% methanol, and F represents PcZ-1grown in the presence of 10 μM propiconazole. Growth of the wild typeChlorella sorokiniana is inhibited in the presence of propiconazole andtypically the culture crashes after a few days. The propiconazoleresistant mutants of Chlorella species (PcZ-1) was not affected by thepresence of propiconazole, and continued to grow. Further, some of themresulted in 10% to 30% higher biomass in the absence of propiconazole ascompared to the wild type progenitor strain. The percent increase ingrowth of PcZ-1 calculated from FIG. 1, is summarized in Table-2.

TABLE 2 Comparison of Wild type Chlorella sorokiniana withoutpropiconazole and PcZ-1 with and without 10 μM propiconazole % increaseAverage % increase No. of PcZ-1 + 10 μM PcZ-1 + 10 days PcZ-1 PcZ PcZ-1uM PcZ 4 54.5 48.1 47.6 45.5 66.7 68.6 50.1 60.2 6 16.3 46.0 26.8 17.338.7 25.4 29.7 27.1 7 7.4 20.0 17.6 10.6 29.4 23.2 15.0 21.1

It is clearly seen from FIG. 1 and Table-2 that the propiconazoleresistant mutants of Chlorella species of the present disclosure showhigher biomass productivity as compared to that of the wild type in thepresent of propiconazole. The higher biomass of the propiconazoleresistant mutants of Chlorella sp. CCAP 211/133 may be due to theenhanced Brassinosteroid (BR) production by the mutant strains. BR's aresteroidal plant hormones that play important roles in plants inregulating cell growth, division as described above.

TECHNICAL ADVANCEMENTS

The present disclosure described herein above has several technicaladvantages including, but not limited to, the realization of:

-   -   propiconazole resistant mutants of Chlorella sp.;    -   a method for preparing propiconazole resistant mutants of        Chlorella sp.;    -   a method for preparing mutants having increased biomass        capability using propiconazole selection;    -   outdoor cultivation of algae without culture crashes due to        contamination by grazers and contaminating organisms; and    -   large scale production of algae devoid of any contamination from        grazers and contaminating organisms.

The embodiments as described herein above, and various features andadvantageous details thereof are explained with reference to thenon-limiting embodiments in the description. Descriptions of well-knownaspects, components and molecular biology techniques are omitted so asto not unnecessarily obscure the embodiments herein.

The foregoing description of specific embodiments so fully reveal thegeneral nature of the embodiments herein, that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationsof such specific embodiments without departing from the generic concept,and, therefore, such adaptations and modifications should and areintended to be comprehended within the meaning and range of equivalentsof the disclosed embodiments. It is to be understood that thephraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein can bepracticed with modification within the spirit and scope of theembodiments as described herein. Further, it is to be distinctlyunderstood that the foregoing descriptive matter is to be interpretedmerely as illustrative of the disclosure and not as a limitation.

Having described and illustrated the principles of the presentdisclosure with reference to the described embodiments, it will berecognized that the described embodiments can be modified in arrangementand detail without departing from the scope of such principles.

While considerable emphasis has been placed herein on the particularfeatures of this disclosure, it will be appreciated that variousmodifications can be made, and that many changes can be made in thepreferred embodiment without departing from the principles of thedisclosure. These and other modifications in the nature of thedisclosure or the preferred embodiments will be apparent to thoseskilled in the art from the disclosure herein, whereby it is to bedistinctly understood that the foregoing descriptive matter is to beinterpreted merely as illustrative of the disclosure and not as alimitation.

We claim:
 1. A propiconazole resistant mutant strain of Chlorellasorokiniana having the Culture Collection of Algae and Protozoa (CCAP)Accession number CCAP 211/133.
 2. A method for selectively controllingthe growth of grazers in an aquatic environment; said method comprisingthe following steps: a. inoculating said aquatic environment with thepropiconazole resistant mutant strain of Chlorella sorokiniana havingAccession No. CCAP 211/133 of claim 1; and b. treating said aquaticenvironment either before or after step a) or both before and after stepa) with an effective concentration of propiconazole to selectivelycontrol the growth of grazers.
 3. The method as claimed in claim 2,wherein the concentration of said propiconazole is in the range of 5 μMto 15 μM.